Eukaryotes existed exclusively as microorganisms for a minimum of 500,000 to one billion years prior to the evolution of plants, animals and fungi. Understanding the evolutionary history of microbial eukaryotes, especially the protists, is key to understanding the origins of eukaryotes, the emergence of parasitism, and the evolution of this remarkably diverse assemblage into plants, animals, fungi and other recently derived groups. Protists represent an eclectic group of organisms marked by numerous innovations in body plans, ecology, biochemistry, and molecular processes. The transition from prokaryotic life-forms to eukaryotic cellular architecture, and the origins of multi-cellular eukaryotes are rooted in the world of protists. [unreadable] [unreadable] This proposal describes the collection of new molecular data and the construction of a large-scale, multi-gene data set for exploring evolutionary relationships for diverse eukaryotes. Our experimental strategy seeks to capture the diversity of eukaryotes by exploring relationships within major clades, and by placing taxa of uncertain position on genealogies. We will construct normalized cDNA libraries for as many as 30 key protists and sequence 10,000-20,000 cDNA clones from each library. Using computer-generated alignments we will use high-throughput phylogenomics to address the following questions: What is the composition and relative branching order of major eukaryotic lineages? To what extent has lateral gene transfer affected the evolution of eukaryotic genomes? Is there a genomic core that is refractory to horizontal gene transfer? Does the pattern of metabolic evolution mirror environmental changes in earth's history? Does adaptation to parasitic life-styles impose major changes in genome architecture and its transcriptome? And finally, what are the key ultrastructural and morphological innovations that led to modem eukaryotic complexity? These phylogenetic frameworks generated by the project will become the basis for interpreting the origin and evolution of genes and genome organization patterns that are responsible for novel phenotypes that underpin organismal complexity and disease. [unreadable] [unreadable]